CAJ | 학술논문

针对~(18)O同位素标记反应两个重要影响因素--肽段分散度和胰酶灭活方法,进行了标记条件的改进和灭活方法的优化.在H_2~(18)O中加入Rapigest~(TM) SF助溶剂并微波辅助加热,使α-酪蛋白胰酶酶切肽段的标记效率得到明显改进(~(18)O/~(16)O峰面积比值>99%).标记后,对胰酶进行还原烷基化化学修饰彻底灭活,使标记后的肽段稳定性显著提高,放置6 d不发生回交反应.对标准蛋白质甲状腺球蛋白酶切肽段混合物标记后的质谱实验结果表明:优化的标记方法能快速稳定地标记蛋白质酶切多肽.
침대~(18)O동위소표기반응량개중요영향인소--태단분산도화이매멸활방법,진행료표기조건적개진화멸활방법적우화.재H_2~(18)O중가입Rapigest~(TM) SF조용제병미파보조가열,사α-락단백이매매절태단적표기효솔득도명현개진(~(18)O/~(16)O봉면적비치>99%).표기후,대이매진행환원완기화화학수식철저멸활,사표기후적태단은정성현저제고,방치6 d불발생회교반응.대표준단백질갑상선구단백매절태단혼합물표기후적질보실험결과표명:우화적표기방법능쾌속은정지표기단백질매절다태.
In order to optimize the ~(18)O labeling method, two key aspects, peptide dispersion and trypsin deac tivation were discussed o The addition of Rapigest SF in H_2~('8)O and microwave heating enhanced labeling efficiency of α-casein digested peptides(~(18)O/~(16)O) ratio >99%).Chemical modification with tris(2-carboxyeth yl) phosphine (TCEP) and iodoacetamide (IAA) resulted in trypsin deactivated completely.No significant back-exchange from ~(18)O to ~(16)O was observed after labeling in 6 days.The experiment result with peptide mixture from showed that the improved method could be effectively used to label protein and peptide.